The determination of creatine kinase (CK) or CK isoenzyme levels in human serum is important in diagnosing a number of physiological conditions. An increased level of CK can be indicative of myocardial infarction, myocardial ischemia, stenocardia, tachycardia, myocarditis, subarachnoid hemorrage, stroke, brain tumor, meningitis and encephalitis, among other conditions.
Three predominant isoenzymes of CK are recognized; these are dimers consisting of the M and B sub-units. The predominant dimer present in the blood, serum or plasma of normal individuals is CK-MM isoenzyme, with variable but usually trace quantities of CK-MB that indicate the normal degradation of skeletal muscle. The CK-BB isoenzyme is not usually present in detectable amounts in serum of normal individuals but is present in significant quantities in brain tissue and smooth muscle tissue.
Elevations of the CK-BB isoenzyme can occur in physiological conditions such as metastatic carcinoma or severe burns. Elevated levels of CK-MB isoenzyme have been used as an indicator of myocardial infarction where possible sources of significant skeletal muscle damage can be eliminated. More particularly, repetitive determinations of CK-MB levels in serum can indicate the time course and severity of infarctions.
More recently, the isoforms of CK isoenzymes have received attention as potential markers for early diagnosis of myocardial infarction. There are three isoforms of CK-MM isoenzyme; CK-MM1, CK-MM2 and CK-MM3; and two isoforms of CK-MB isoenzyme; CK-MB1 and CK-MB2. Shortly after myocardial infarction, the ratio of CK-MM and CK-MB isoforms contained in tissue to that contained in plasma increases. Such increase occurs several hours before either total CK or CK-MB activity exceeds normal reference values.
A number of diagnostic assays have been developed for the determination of the presence or the concentration of CK or CK isoenzymes in human serum. Many such assays are based on measurement of the enzymatic activity of the CK enzyme or isoenzyme. Difficulties in measuring the enzymatic activity of the CK-MB isoenzyme and the need for greater sensitivity have led to the development of immunochemical mass determinations for CK-MB. Such assays are highly dependent on the immunoreactivity of the CK-MB isoenzyme.
It has been observed that CK and CK isoenzymes are highly unstable. CK and CK isoenzymes are sulfhydryl-requiring, that is, they need to maintain their --SH groups in their free, unoxidized form. Atmospheric oxygen causes the oxidation of the --SH groups to the disulfide linkage, thereby diminishing enzyme stability and catalytic activity. Additionally, changes to the CK and CK isoenzyme molecules caused by increased temperatures and the presence of proteases and reducing agents can change the immunoreactivity of the molecules.
Storage stability of patient serum samples containing CK and CK isoenzymes is important, especially when further analysis on a sample is requested retrospectively following an equivocal ECG or when the cause of a raised total CK level is being sought. Such a request may be made the same day or days after the initial ECG or CK assay was performed. Buttery et al. [Clinical Biochemistry, 25: 11-13 (1992)] report that samples stored at -20.degree. C. overnight show, on average, a decrease in CK-MB isoenzyme values of 13.5%. Such a decrease could result in a misdiagnosis. Prolonged storage of samples at room temperature for 4-6 days showed a 50% deterioration of CK-MB when measured electrophoretically and 20% deterioration when measured by an immunochemical mass assay. Therefore, a need exists for a means for stabilizing CK and CK isoenzyme activity in patient serum samples.
CK and CK isoenzyme assay control products containing CK or CK isoenzyme also suffer from instability problems. In order to facilitate storage of such control products, they are typically lyophilized. A useful lyophilized CK or CK isoenzyme control product must have two characteristics which are often very difficult to achieve simultaneously. First, there must be substantially no or only very small temperature of hydration effect and, second, there must only be very slight changes in enzyme activity or immunoreactivity upon standing after reconstitution (rehydration) of the lyophilized control product. The temperature of hydration effect is the phenomenon of variability of recovered enzyme activity and immunoreactivity from a lyophilized control product upon rehydration caused by the differences in the temperature of water used. For practical purposes, for use in diagnostic assays, lyophilized control products must have a small temperature of hydration effect to permit reproducible analytical results.
Thiols, such as N-acetyl cysteine, glutathione and monothioglycerol, have been used as additives to lyophilized CK and CK isoenzyme control products as thiol protectors. (See U.S. Pat. No. 4,442,212, issued Apr. 10, 1984 to Briggs.) While use of such thiols in lyophilized CK and CK isoenzyme control products has been found to control the stability of CK and CK isoenzyme enzymatic activity after reconstitution, use of thiols has little effect on stability of CK and CK isoenzyme immunoreactivity.
U.S. Pat. No. 4,994,375, issued Feb. 19, 1991 to Posner et al., discloses a stable reconstituted human serum-based control for the assay of total lactate dehydrogenase (LDH) and CK and their isoenzymes which is comprised of human serum, LDH and LDH isoenzymes, CK and CK isoenzymes and sodium citrate. The pH of the control product is adjusted to 6.7 before lyophilization. In this control product formulation, there is no means disclosed for maintaining the pH of the control product at 6.7 after rehydration. Variability in pH of the control product after reformulation can lead to inconsistent results. Additionally, there is no suggestion that the disclosed control product formulation would stabilize immunoreactivity of CK or CK isoenzymes.
There is a need for a formulation for stabilizing enzymatic activity and immunoreactivity of CK and CK isoenzymes which can be used to stabilize human serum samples containing CK and CK isoenzymes and as a reconstitutable lyophilized CK and CK isoenzyme control product which exhibits little temperature of hydration effect and which is stable after reconstitution.